ATF5 Peptide Variants and Uses Thereof

ABSTRACT

Provided are ATF5 peptides having a truncated ATF5 leucine zipper region and, optionally, a cell-penetrating region, compositions comprising the ATF5 peptides, and methods of inhibiting proliferation of and promoting cytotoxicity in a neoplastic cell using the ATF5 peptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/613,083, filed on Jan. 3, 2018.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 21, 2018, isnamed Sapience_003_US1_SL.txt and is 29,385 bytes in size.

BACKGROUND

Activating transcription factor 5 (ATF5) is a member of the ATF/CREB(cAMP response element binding protein) family of basic leucine zipperproteins. In the normal developing brain, ATF5 is highly expressed inneural progenitor/neural stem cells where it blocks cell cycle exit andpromotes cell proliferation, thereby inhibiting neurogenesis andgliogenesis. ATF5 downregulation is required to permit neuroprogenitorcell cycle exit and differentiation into either neurons, astrocytes, oroligodendroglia (Greene et al. 2009; Sheng et al. 2010a; Sheng et al.2010b; Arias et al. 2012).

In addition to its role in normal development of the nervous system,ATF5 has also emerged as an oncogenic factor that promotes survival ofgliomas and other tumors. A number of studies have demonstrated thatATF5 is highly expressed in a variety of cancers, includingglioblastoma, breast, pancreatic, lung, and colon cancers, and isessential for glioma cell survival (Monaco et al. 2007; Sheng et al.2010a). In the context of gliomas, overexpression of ATF5 inverselycorrelates with disease prognosis and survival, i.e., glioma patientswith higher ATF5 expression have significantly worse outcomes thanpatients with lower ATF5 expression.

In cancer cells, genes that induce apoptosis are often inactivated ordown-regulated, whereas anti-apoptotic genes are frequently activated oroverexpressed. Consistent with this paradigm, ATF5 upregulatestranscription of anti-apoptotic proteins, including B-cell leukemia 2(Bcl-2) and myeloid cell leukemia 1 (Mcl-1), promoting tumor cellsurvival (Sheng et al., 2010b; Chen et al., 2012).

SUMMARY OF THE INVENTION

Some of the main aspects of the present invention are summarized below.

Additional aspects are described in the Detailed Description of theInvention, Examples, Drawings, and Claims sections of this disclosure.The description in each section of this disclosure is intended to beread in conjunction with the other sections. Furthermore, the variousembodiments described in each section of this disclosure can be combinedin various different ways, and all such combinations are intended tofall within the scope of the present invention.

Accordingly, the disclosure provides peptide derivatives of ATF5comprising an ATF5 leucine zipper domain, optionally wherein anengineered enhanced leucine zipper sequence is absent, compositions andkits comprising the ATF5 peptides, and methods for inducing cytotoxicityin and/or inhibiting proliferation of a neoplastic cell using the ATF5peptides. In particular, the disclosure provides variants of an ATF5peptide comprising the ST-3 leucine zipper sequence (SEQ ID NO: 53),which variants can comprise non-conservative amino acid substitutions.Prior to the present invention, it could not have been predicted thatsuch substitutions would result in molecules having similar or superiorcytotoxic activity, compared with an ATF5 peptide comprising the ST-3leucine zipper sequence (SEQ ID NO: 53). In some embodiments, thedisclosure provides variants of the cell-penetrating ATF5 peptide ST-3,which variants have similar or superior cytotoxic activity, comparedwith ST-3.

In one aspect, the invention provides an ATF5 peptide comprising atruncated ATF5 leucine zipper region, wherein the truncated ATF5 leucinezipper region comprises a variant of the amino acid sequenceLEGECQGLEARNRELKERAESV (SEQ ID NO: 53), wherein the variant is modifiedat one or more positions of SEQ ID NO: 53 as follows: (i) E4 issubstituted with a positively charged residue; (ii) C5 is substitutedwith a non-polar residue; (iii) Q6 is substituted with alanine; (iv) E9is substituted with a positively charged residue; (v) R11 is substitutedwith a negatively charged residue; (vi) N12 is substituted with anon-polar residue; (vii) K16 is substituted with a negatively chargedresidue; (viii) S21 is substituted with alanine.

In another aspect, the invention provides an ATF5 peptide comprising atruncated ATF5 leucine zipper region, wherein the truncated ATF5 leucinezipper region comprises an amino acid sequence selected from the groupconsisting of:

(SEQ ID NO: 54) LEGEGQGLEARNRELKERAESV, (SEQ ID NO: 55)LEGEAQGLEARNRELKERAESV, (SEQ ID NO: 56) LEGECQGLEARNRELKERAEAV(SEQ ID NO: 57) LEGECQGLEARLRELKERAESV, (SEQ ID NO: 58)LEGECAGLEARNRELKERAESV, (SEQ ID NO: 59) LEGRCQGLRAENRELEERAESV,(SEQ ID NO: 60) LEGRCQGLRAELRELEERAEAV, and (SEQ ID NO: 61)LEGRAQGLRAELRELEERAEAV.

In one embodiment, the ATF5 peptide further comprises a cell-penetratingregion, such that the ATF5 peptide is a cell-penetrating peptide. Insome embodiments, the cell-penetrating region has an amino acid sequenceselected from the group consisting of:

(SEQ ID NO: 25) RQIKIWFQNRRMKWKK, (SEQ ID NO: 26) RQLKLWFQNRRMKWKK,(SEQ ID NO: 40) YGRKKRRQRRR, and (SEQ ID NO: 41) YGRKKRRQRR.

A further aspect of the invention provides a cell-penetrating ATF5peptide comprising a variant of the amino acid sequenceRQIKIWFQNRRMKWKKLEGECQGLEARNRELKERAESV (SEQ ID NO: 3), wherein thevariant is modified at positions of SEQ ID NO: 3 selected from the groupconsisting of: (i) C21G (SEQ ID NO: 4); (ii) C21A (SEQ ID NO: 5); (iii)Q22A (SEQ ID NO: 8); (iv) E20R, E25R, R27E, and K32E (SEQ ID NO: 9); (v)N28L (SEQ ID NO: 7); (vi) S37A (SEQ ID NO: 6); (vii) E20R, E25R, R27E,N28L, K32E, and S37A (SEQ ID NO: 10); and (viii) E20R, C21A, E25R, R27E,N28L, K32E, and S37A (SEQ ID NO: 11).

In certain embodiments, the ATF5 peptide comprises D-amino acids, in areversed amino acid sequence relative to an L-amino acid sequence of anATF5 peptide of the invention. In one embodiment, the truncated ATF5leucine zipper region of the retro inverso peptide has a D-amino acidsequence VAEAREELERLEARLGQARGEL (SEQ ID NO: 65). In a particularembodiment, the retro inverso peptide is a cell-penetrating ATF5 peptidecomprising a D-amino acid sequence

(SEQ ID NO: 14) VAEAREELERLEARLGQARGELKKWKMRRNQFWLKLQR.

In some embodiments, the ATF5 peptide of the invention does not comprisean extended leucine zipper region.

In some embodiments, the ATF5 peptide of the invention comprises anN-terminal acetyl group and/or a C-terminal amide group.

Also provided is a composition comprising an ATF5 peptide of theinvention. In some embodiments, the composition is a pharmaceuticalcomposition. Further provided is a kit comprising an ATF5 peptide of theinvention, and a nucleic acid molecule encoding an ATF5 peptide of theinvention.

The invention provides an ATF5 peptide of the invention for use inpromoting cytotoxicity in a neoplastic cell. The invention furtherprovides an ATF5 peptide of the invention for use in inhibitingproliferation of a neoplastic cell. The invention additionally providesa method of promoting cytotoxicity in a neoplastic cell, the methodcomprising contacting the neoplastic cell with an ATF5 peptide of theinvention. The invention additionally provides a method of inhibitingproliferation in a neoplastic cell, the method comprising contacting theneoplastic cell with an ATF5 peptide of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C show amino acid sequences of the ATF-5 peptides NTAzip-ATF5(SEQ ID NO: 1) (FIG. 1A), ST-2 (SEQ ID NO: 2) (FIG. 1B), and ST-3 (SEQID NO: 3) (FIG. 1C). The extended leucine zipper domain is underlined,the Penetratin cell-penetrating domain is italicized, and the ATF-5leucine zipper domain is bolded.

FIG. 2 shows in vitro activity of ST-3 variants in which the singlecysteine is replaced.

FIG. 3 shows in vitro activity of ST-3 variants in which single aminoacid substitutions have been made.

FIG. 4 shows in vitro activity of ST-3 variants in which multiple aminoacid substitutions have been made.

FIG. 5 shows in vitro activity of ST-13, a retro inverso version ofST-11, an ST-3 variant.

FIGS. 6A-6E show in vitro activity of ST-13 versus ST-3 in HL60 humanpromyelocytic leukemia cells (PML) (FIG. 6A), acute myeloid leukemiacells (AML14 and SET2) (FIGS. 6B, 6C), melanoma cells (A375) (FIG. 6D),and breast cancer cells (MCF7) (FIG. 6E). EC₅₀ values in each cell typefor ST-3 and ST-13, respectively, were 19.0 μM and 4.8 μM (FIG. 6A);29.6 μM and <1 (FIG. 6B); 98.2 μM and 17.7 (FIG. 6C); 21.8 μM and 1.4 μM(FIG. 6D); and 52.9 μM and <1 (FIG. 6E).

FIGS. 7A-7E show in vitro activity of ST-14 in HL60 human promyelocyticleukemia cells (PML) (FIG. 7A), acute myeloid leukemia cells (AML14)(FIG. 7B), glioblastoma cells (U251) (FIG. 7C), melanoma cells (A375)(FIG. 7D), and breast cancer cells (MCF7) (FIG. 7E). EC₅₀ value for ST-2and ST-14, respectively, were >300 and <5 μM (FIG. 7A).

FIGS. 8A-8D show that treatment with ST-14 downregulates expression ofMcl-1, Bcl-2, BIRC5 (Survivin), and ATF5. RNA expression was measured byreverse transcription polymerase chain reaction (RT-PCR). Expressionlevels in HL60 cells treated with 5 μM ST-14 are shown relative to(3-actin expression at 4 hours (FIG. 8A) and 24 hours (FIG. 8B)post-treatment. Expression levels in U251 cells (FIG. 8C) and HL60 cells(FIG. 8D) treated with 0, 20, or 40 μM ST-14 are shown relative to(3-actin expression after 4 hours (FIG. 8C) or 24 hours (FIG. 8D) oftreatment.

FIG. 9 shows the anti-tumor activity of ST-3 variants in an HL60subcutaneous tumor model. Nu/J mice were treated with 25 mg/kg BID-IP(n=6-7 per group).

FIGS. 10A-10C show anti-tumor activity of ST-14 in a U251 subcutaneoustumor model. NOD/SCID mice were treated with 50 mg/kg SC three times perweek for three weeks. Average starting tumor volume was about 240 mm³.Mean tumor volume (FIG. 10A), percent survival (FIG. 10B), andindividual tumor volumes (FIG. 10C) are shown. Data points representmean±SEM.; p<0.0001; n=number of live animals per group at eachtimepoint.

FIGS. 11A-11B show that early or delayed ST-14 administration hassignificant anti-tumor activity in MCF7 breast cancer cells. Nu/J micewere treated with 25 mg/kg SC three times per week for three weeksbeginning 2 days (FIG. 11A) or 59 days (FIG. 11B) post tumorinoculation. Average starting tumor volume was about 280-330 mm³.Inoculation: 2×10⁶ cells in Vehicle groups (FIGS. 11A, 11B); 2×10⁶ cellsin ST-14 group (FIG. 11A); 5×10⁶ cells in ST-14 group (FIG. 11B). Datapoints represent mean±SEM.; p<0.0001.

FIG. 12 shows anti-tumor activity of ST-14 in an HL60 subcutaneous tumormodel. Nu/J mice were treated with 20 mg/kg SC three times per week forthree weeks. Average starting tumor volume was about 220 mm³. Datapoints represent mean±SEM; p<0.05.

FIG. 13 shows anti-tumor activity of ST-14 in a A375 subcutaneous tumormodel. NOD/SCID mice were treated with 25 mg/kg SC twice daily for threeweeks. Average starting tumor volume was about 250-344 mm³. Data pointsrepresent mean±SEM; p=0.002.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of pharmaceutics, formulationscience, protein chemistry, cell biology, cell culture, molecularbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art.

In order that the present invention can be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the disclosure. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionis related.

Any headings provided herein are not limitations of the various aspectsor embodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

All references cited in this disclosure are hereby incorporated byreference in their entireties. In addition, any manufacturers'instructions or catalogues for any products cited or mentioned hereinare incorporated by reference. Documents incorporated by reference intothis text, or any teachings therein, can be used in the practice of thepresent invention. Documents incorporated by reference into this textare not admitted to be prior art.

I. Definitions

The phraseology or terminology in this disclosure is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents, unless the contextclearly dictates otherwise. The terms “a” (or “an”) as well as the terms“one or more” and “at least one” can be used interchangeably.

Furthermore, “and/or” is to be taken as specific disclosure of each ofthe two specified features or components with or without the other.Thus, the term “and/or” as used in a phrase such as “A and/or B” isintended to include A and B, A or B, A (alone), and B (alone). Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to include A, B, and C; A, B, or C; A or B; A or C; B or C; Aand B; A and C; B and C; A (alone); B (alone); and C (alone).

Wherever embodiments are described with the language “comprising,”otherwise analogous embodiments described in terms of “consisting of”and/or “consisting essentially of” are included.

Units, prefixes, and symbols are denoted in their Systéme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range, and any individual value provided herein canserve as an endpoint for a range that includes other individual valuesprovided herein. For example, a set of values such as 1, 2, 3, 8, 9, and10 is also a disclosure of a range of numbers from 1-10, from 1-8, from3-9, and so forth. Likewise, a disclosed range is a disclosure of eachindividual value encompassed by the range. For example, a stated rangeof 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength, and their salts. The polymer can be linear or branched, cancomprise modified amino acids, and can be interrupted by non-aminoacids. Except where indicated otherwise, e.g., for the abbreviations forthe uncommon or unnatural amino acids set forth herein, the three-letterand one-letter abbreviations, as used in the art, are used herein torepresent amino acid residues. Except when preceded with a “D” or inlower case, the amino acid is an L-amino acid. Groups or strings ofamino acid abbreviations are used to represent peptides. Except wherespecifically indicated, peptides are indicated with the N-terminus ofthe left and the sequence is written from the N-terminus to theC-terminus.

The terms “polypeptide,” “peptide,” and “protein” also encompass anamino acid polymer that has been modified naturally or by intervention;for example, disulfide bond formation, lactam bridge formation,glycosylation, lipidation, acetylation, acylation, amidation,phosphorylation, or other manipulation or modification, such asconjugation with a labeling component or addition of a protecting group.Also included within the definition are, for example, polypeptidescontaining one or more analogs of an amino acid (including, for example,amino-isobutyric acid (Aib), unnatural amino acids, etc.) andpolypeptides comprising or consisting of D-amino acids, as well as othermodifications known in the art. In certain embodiments, the polypeptidescan occur as single chains, covalent dimers, or non-covalent associatedchains. Polypeptides can also be in cyclic form. Cyclic polypeptides canbe prepared, for example, by bridging free amino and free carboxylgroups. Formation of the cyclic compounds can be achieved by treatmentwith a dehydrating agent, with suitable protection if needed. The openchain (linear form) to cyclic form reaction can involveintramolecular-cyclization. Cyclic polypeptides can also be prepared byother methods known in the art, for example, using one or more lactambridges, hydrogen bond surrogates (Patgiri et al. 2008), hydrocarbonstaples (Schafmeister et al. 2000), triazole staples (Le Chevalier Isaadet al. 2009), or disulfide bridges (Wang et al. 2006). Bridges orstaples can be spaced, for example, 3, 4, 7, or 8 amino acids apart.

The term “variant” refers to a peptide having one or more amino acidsubstitutions, deletions, and/or insertions compared to a referencesequence. Deletions and insertions can be internal and/or at one or moretermini. Substitution can include the replacement of one or more aminoacids with a similar or homologous amino acid(s) or a dissimilar aminoacid(s). For example, some variants include alanine substitutions at oneor more amino acid positions. Other substitutions include conservativesubstitutions that have little or no effect on the overall net charge,polarity, or hydrophobicity of the protein. Some variants includenon-conservative substitutions that change the charge or polarity of theamino acid. Substitution can be with either the L- or the D-form of anamino acid.

A “retro inverso” peptide has a reversed amino acid sequence, relativeto a reference L-amino acid sequence, and is made up of D-amino acids(inverting the α-center chirality of the amino acid subunits) to helpmaintain side-chain topology similar to that of the original L-aminoacid peptide.

The term “conservative substitution” as used herein denotes that one ormore amino acids are replaced by another, biologically similar residue.Examples include substitution of amino acid residues with similarcharacteristics, e.g., small amino acids, acidic amino acids, polaramino acids, basic amino acids, hydrophobic amino acids, and aromaticamino acids. For further information concerning phenotypically silentsubstitutions in peptides and proteins, see, for example, Bowie et. al.,Science 247:1306-1310 (1990). In the scheme below, conservativesubstitutions of amino acids are grouped by physicochemical properties;I: neutral and/or hydrophilic, II: acids and amides, III: basic, IV:hydrophobic, V: aromatic, bulky amino acids.

I II III IV V A N H M F S D R L Y T E K I W P Q V G C

In the scheme below, conservative substitutions of amino acids aregrouped by physicochemical properties; VI: neutral or hydrophobic, VII:acidic, VIII: basic, IX: polar, X: aromatic.

VI VII VIII IX X A D H M F L E R S Y I K T W V N H P Q G C

Methods of identifying conservative nucleotide and amino acidsubstitutions which do not affect protein function are well-known in theart (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashiet al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc.Natl. Acad. Sci. U.S.A. 94:412-417 (1997)).

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or peptides, refers to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms, or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences.

One such non-limiting example of a sequence alignment algorithm isdescribed in Karlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990),as modified in Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877(1993), and incorporated into the NBLAST and XBLAST programs (Altschulet al., Nucleic Acids Res., 25:3389-3402 (1991)). In certainembodiments, Gapped BLAST can be used as described in Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul etal., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2(Genentech, South San Francisco, Calif.) or Megalign (DNASTAR) areadditional publicly available software programs that can be used toalign sequences. In certain embodiments, the percent identity betweentwo nucleotide sequences is determined using the GAP program in the GCGsoftware package (e.g., using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). Incertain alternative embodiments, the GAP program in the GCG softwarepackage, which incorporates the algorithm of Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)), can be used to determine the percentidentity between two amino acid sequences (e.g., using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certainembodiments, the percent identity between nucleotide or amino acidsequences is determined using the algorithm of Myers and Miller (CABIOS4:11-17 (1989)). For example, the percent identity can be determinedusing the ALIGN program (version 2.0) and using a PAM120 with residuetable, a gap length penalty of 12 and a gap penalty of 4. One skilled inthe art can determine appropriate parameters for maximal alignment byparticular alignment software. In certain embodiments, the defaultparameters of the alignment software are used. Other resources forcalculating identity include methods described in ComputationalMolecular Biology (Lesk ed., 1988); Biocomputing: Informatics and GenomeProjects (Smith ed., 1993); Computer Analysis of Sequence Data, Part 1(Griffin and Griffin eds., 1994); Sequence Analysis in Molecular Biology(G. von Heinje, 1987); Sequence Analysis Primer (Gribskov et al. eds.,1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988).

A “polynucleotide,” as used herein can include one or more “nucleicacids,” “nucleic acid molecules,” or “nucleic acid sequences,” andrefers to a polymer of nucleotides of any length, and includes DNA andRNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase. Apolynucleotide can comprise modified nucleotides, such as methylatednucleotides and their analogs. The preceding description applies to allpolynucleotides referred to herein, including RNA and DNA.

An “isolated” molecule is one that is in a form not found in nature,including those which have been purified.

A “label” is a detectable compound that can be conjugated directly orindirectly to a molecule, so as to generate a “labeled” molecule. Thelabel can be detectable on its own (e.g., radioisotope labels orfluorescent labels), or can be indirectly detected, for example, bycatalyzing chemical alteration of a substrate compound or compositionthat is detectable (e.g., an enzymatic label) or by other means ofindirect detection (e.g., biotinylation).

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a moleculeand its binding partner (e.g., a receptor and its ligand, an antibodyand its antigen, two monomers that form a dimer, etc.). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair. The affinity of a molecule X for its partner Y cangenerally be represented by the dissociation constant (K_(D)). Affinitycan be measured by common methods known in the art, including thosedescribed herein. Low-affinity binding partners generally bind slowlyand tend to dissociate readily, whereas high-affinity binding partnersgenerally bind faster and tend to remain bound longer.

The affinity or avidity of a molecule for its binding partner can bedetermined experimentally using any suitable method known in the art,e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), orradioimmunoassay (RIA), or kinetics (e.g., KINEXA® or BIACORE™ or OCTET®analysis). Direct binding assays as well as competitive binding assayformats can be readily employed. (See, e.g., Berzofsky et al.,“Antibody-Antigen Interactions,” in Fundamental Immunology, Paul, W. E.,ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W.H. Freemanand Company: New York, N.Y. (1992)). The measured affinity of aparticular binding pair interaction can vary if measured under differentconditions (e.g., salt concentration, pH, temperature). Thus,measurements of affinity and other binding parameters (e.g., K_(D) orK_(d), K_(on), K_(off)) are made with standardized solutions of bindingpartners and a standardized buffer, as known in the art.

An “active agent” is an ingredient that is intended to furnishbiological activity. The active agent can be in association with one ormore other ingredients. An active agent that is a peptide can also bereferred to as an “active peptide.”

An “effective amount” of an active agent is an amount sufficient tocarry out a specifically stated purpose.

The term “pharmaceutical composition” refers to a preparation that is insuch form as to permit the biological activity of the active ingredientto be effective and which contains no additional components that areunacceptably toxic to a subject to which the composition would beadministered. Such composition can be sterile and can comprise apharmaceutically acceptable carrier, such as physiological saline.Suitable pharmaceutical compositions can comprise one or more of abuffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g.polysorbate), a stabilizing agent (e.g. polyol or amino acid), apreservative (e.g. sodium benzoate), and/or other conventionalsolubilizing or dispersing agents.

A “subject” or “individual” or “animal” or “patient” or “mammal,” is anysubject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, sports animals, and laboratory animalsincluding, e.g., humans, non-human primates, canines, felines, porcines,bovines, equines, rodents, including rats and mice, rabbits, etc.

The terms “inhibit,” “block,” and “suppress” are used interchangeablyand refer to any statistically significant decrease in occurrence oractivity, including full blocking of the occurrence or activity. Forexample, “inhibition” can refer to a decrease of about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An“inhibitor” is a molecule, factor, or substance that produces astatistically significant decrease in the occurrence or activity of aprocess, pathway, or molecule.

A “neoplastic cell” or “neoplasm” typically has undergone some form ofmutation/transformation, resulting in abnormal growth as compared tonormal cells or tissue of the same type. Neoplasms include morphologicalirregularities, as well as pathologic proliferation. Neoplastic cellscan be benign or malignant. Malignant neoplasms, i.e., cancers, aredistinguished from benign in that they demonstrate loss ofdifferentiation and orientation of cells, and have the properties ofinvasion and metastasis.

A “solid tumor” is a mass of neoplastic cells. A “liquid tumor” or a“hematological malignancy” is a blood cancer of myeloid or lymphoidlineage.

II. ATF5 Peptides and Compositions ATF5 Peptides

ATF5 is a 282-amino acid eukaryotic transcription factor with anN-terminal acidic activation domain and a C-terminal basic leucinezipper (bZIP) domain. The bZIP domain contains a DNA-binding region anda leucine zipper region. The leucine zipper is a common structuralmotif, typically having a leucine at every seventh amino acid in thedimerization domain. bZIP transcription factors homo- and/orhetero-dimerize via their leucine zippers to specifically bind to DNA.Wild-type human, rat, and murine ATF5 have the amino acid sequences setforth in NCBI Accession No. NP_001180575, NP_758839, and NP_109618,respectively.

NTAzip-ATF5 (FIG. 1A) is an ATF5 peptide in which the ATF5 N-terminalactivation domain is deleted and the DNA binding domain is replaced withan engineered enhanced leucine zipper, i.e., an amphipathic acidicα-helical sequence containing heptad repeats with a leucine at everyseventh residue, which extends the wild-type ATF5 leucine zipper region(Angelastro et al. 2003). Cell-penetrating dominant negative ATF5(CP-d/n-ATF5) molecules are improved versions of NTAzip-ATF5, whichcontain a cell-penetrating domain and a truncated ATF5 leucine zippersequence (relative to wild-type), along with an extended leucine zippersequence (US 2016/0046686; Karpel-Massler et al. 2016). An example of aCP-d/n ATF5 molecule is shown in FIG. 1B. As used herein, the terms“extended leucine zipper,” “leucine zipper extension,” and “enhancedleucine zipper” refer to a peptide having from one to four leucineheptads, i.e., Leu-(X)₆ (SEQ ID NO: 52), which sequence is not awild-type ATF5 leucine zipper sequence.

ST-3 (FIG. 1C) is a variant of a CP-d/n-ATF5 molecule that lacks theleucine zipper extension, and induces cell death in neoplastic cells.Previous studies demonstrated that the enhanced leucine zipper region isrequired for stability and inhibitory activity of dominant-negative bZIPinhibitors (Krylov et al. 1995; Olive et al. 1997; Moll et al. 2000;Acharya et al. 2006). Therefore, the discovery that ST-3 retains itsability to specifically target and kill neoplastic cells in the absenceof an extended leucine zipper region was unexpected.

The present inventors have discovered that non-conservative variants ofan ATF5 peptide comprising the ST-3 leucine zipper sequence (SEQ ID NO:53) induce cell death in neoplastic cells. The discovery that theATF5-derived peptides of the present invention retain their ability tospecifically target and kill neoplastic cells with multiplenon-conservative amino acid substitutions to the ST-3 leucine zipperregion could not have been predicted prior to the present invention.Retro inverso variants of ST-3 were not only active, but had increasedactivity relative to ST-3, which was unexpected.

The invention provides ATF5 peptides having a truncated ATF5 leucinezipper region and, optionally, a cell-penetrating region. ATF5 peptidesof the invention are capable of interfering with ATF5 activity in a cellinto which they are introduced. In some embodiments, the ATF5 peptidecan affect pathways involved in apoptosis. ATF5 activity can be assessedby any of several assays known in the art, including the cell-killassays described herein. ATF5 activity can also be assessed by itsability to bind to the cAMP-response element (CRE).

The “ATF5 leucine zipper region” is a truncated sequence derived fromthe wild-type ATF5 leucine zipper region. The term is used to refer onlyto sequence, and not necessarily to secondary structure. The truncatedATF5 leucine zipper region can have, for example, an amino acid sequenceshown in Table 1. The ST-3 leucine zipper sequence (SEQ ID NO: 53) isshown as a point of reference. Substitutions in SEQ ID NO: 53 are shownin underlined bold type.

TABLE 1 SEQ ID NO 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 53 L E G E C Q G L E A R N R E L K E R A E S V 54 L E G E G Q G L E AR N R E L K E R A E S V 55 L E G E A Q G L E A R N R E L K E R A E S V56 L E G E C Q G L E A R N R E L K E R A E A V 57 L E G E C Q G L E A RL R E L K E R A E S V 58 L E G E C A G L E A R N R E L K E R A E S V 59L E G R C Q G L R A E N R E L E E R A E S V 60 L E G R C Q G L R A E L RE L E E R A E A V 61 L E G R A Q G L R A E L R E L E E R A E A V 62 L EG R L Q G L R A E L R E L E E R A E A V 63 L E G R L A G L R A E L R E LE E R A E A V 64 L E G R A A G L R A E L R E L E E R A E A V

The leucine zipper region can be a retro inverso form. In oneembodiment, the retro inverso leucine zipper region has the sequenceVAEAREELERLEARLGQARGEL (SEQ ID NO: 65).

Variants of these sequences are also included in the scope of theinvention. ATF5 peptides of the invention can have a leucine zipperregion of at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to those sequences disclosed herein.

In embodiments wherein the ATF5 peptide comprises a cell-penetratingregion, the cell-penetrating region is operably linked to the truncatedATF5 leucine zipper region. In some embodiments, the cell-penetratingregion is covalently linked to the truncated ATF5 leucine zipper region,for example, via a peptide bond, a disulfide bond, a thioether bond, ora linker known in the art (see, e.g., Klein et al. 2014). Exemplarylinkers include, but are not limited to, a substituted alkyl or asubstituted cycloalkyl. Linkers can be cleavable after the peptide isdelivered. A cell-penetrating region and an ATF5 leucine zipper regionlinked directly by an amide bond may be referred to as a “fusion.”Fusions can contain an amino acid linker sequence between thecell-penetrating region and the ATF5 leucine zipper region, as discussedabove with respect to active peptides. The cell-penetrating region canbe linked to the N-terminus or the C-terminus of the truncated ATF5leucine zipper region, or via a residue side chain. The cell-penetratingregion and truncated ATF5 leucine zipper region can have the same oropposite chirality.

Cell-penetrating ATF5 peptides of the invention can comprise anycombination of cell-penetrating and ATF5 leucine zipper domainsdisclosed herein. Non-limiting examples of such peptides are shown inTable 2. The cell-penetrating region is italicized. Substitutionsrelative to the ST-3 sequence are shown in underlined bold type.

TABLE 2 SEQ ID Peptide NO: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 20 ST-3 3 R Q I K I W F Q N R R M K W K K L E G E ST-4 4 R Q I K I WF Q N R R M K W K K L E G E ST-5 5 R Q I K I W F Q N R R M K W K K L E GE ST-6 6 R Q I K I W F Q N R R M K W K K L E G E ST-7 7 R Q I K I W F QN R R M K W K K L E G E ST-8 8 R Q I K I W F Q N R R M K W K K L E G EST-9 9 R Q I K I W F Q N R R M K W K K L E G R ST-10 10 R Q I K I W F QN R R M K W K K L E G R ST-11 11 R Q I K I W F Q N R R M K W K K L E G RST-12 12 R Q I K I W F Q N R R M K W K K L E G R SEQ ID Peptide NO: 2122 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 ST-3 3 C Q G L E A RN R E L K E R A E S V ST-4 4 G Q G L E A R N R E L K E R A E S V ST-5 5A Q G L E A R N R E L K E R A E S V ST-6 6 C Q G L E A R N R E L K E R AE A V ST-7 7 C Q G L E A R L R E L K E R A E S V ST-8 8 C A G L E A R NR E L K E R A E S V ST-9 9 C Q G L R A E N R E L E E R A E S V ST-10 10C Q G L R A E L R E L E E R A E A V ST-11 11 A Q G L R A E L R E L E E RA E A V ST-12 12 A A G L R A E L R E L E E R A E A V

Retro inverso forms of the ATF5 peptides of the invention are alsoincluded. In one embodiment, the ATF5 peptide is ST-13, a retro inversopeptide comprising a cell-penetrating region and having the D-amino acidsequence

(SEQ ID NO: 13) V A EARE E LER LE A R LGQ AR GELKKWKMRRNQFWIKIQR.In another embodiment, the ATF5 peptide is ST-14, a retro inversopeptide comprising a cell-penetrating region and having the D-amino acidsequence

(SEQ ID NO: 14) V A EARE E LER LE A R LGQ AR GELKKWKMRRNQFW

K

QR.The cell-penetrating region is italicized. Substitutions relative to theST-3 sequence (SEQ ID NO: 3) are shown in underlined bold type.

ATF5 peptides of the invention are preferably 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids in length, includingranges having any of those lengths as endpoints, for example, 22-38amino acids.

ATF5 peptides of the invention include peptides having at least about80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to those sequences disclosedherein.

The ATF5 peptides can have a modified N-terminus and/or a modifiedC-terminus. For example, ATF5 peptides can optionally include anN-terminal acetyl group and/or a C-terminal amide group.

ATF5 peptides of the invention can optionally be cyclic. For example,ATF5 peptides can include one or more lactam bridges. A lactam bridge ispreferably, but not necessarily, created between side chains spaced fouramino acid residues apart (BxxxB). Lactam bridges can be formed, forexample, between the side chains of Asp or Glu and Lys. Amino acidsubstitutions can be made at the site of the lactam bridge to facilitatethe linkage.

ATF5 peptides of the invention can optionally include one or moreepitope and/or affinity tags, such as for purification or detection.Non-limiting examples of such tags include FLAG, HA, His, Myc, GST, andthe like. ATF5 peptides of the invention can optionally include one ormore labels.

In certain aspects, the invention provides a composition, e.g., apharmaceutical composition, comprising an ATF5 peptide of the invention,optionally further comprising one or more carriers, diluents,excipients, or other additives.

Also within the scope of the invention are kits comprising the ATF5peptides and compositions as provided herein and, optionally,instructions for use. The kit can further contain at least oneadditional reagent, and/or one or more additional active agent. Kitstypically include a label indicating the intended use of the contents ofthe kit. The term “label” includes any writing or recorded materialsupplied on or with the kit, or that otherwise accompanies the kit.

The ATF5 peptides of the invention promote differential gene expressionof a set of genes including, but not limited to, ATF5 target genesBcl-2, Mcl-1, and Survivin. Specifically, the ATF5 peptides knock-downexpression of ATF5 target genes associated with cell survival,proliferation, and plasticity. Accordingly, the ATF5 peptides can beused to induce cell death, decrease cellular proliferation, or activatecellular differentiation. In certain embodiments, the ATF5 peptides ofthe invention are used to inhibit proliferation of and/or to promotecytotoxicity in a neoplastic cell. Proliferation and cytotoxicity can bemeasured by known assays, including the cell kill assays describedherein.

Cell Targeting

ATF5 peptides of the invention can be introduced into target cells bymethods known in the art. The method of introduction chosen will depend,for example, on the intended application.

In some instances, DNA or RNA encoding the ATF5 peptide can be deliveredto and expressed in a target cell. Delivery can be accomplished via anysuitable vector, depending on the application. Examples of vectorsinclude plasmid, cosmid, phage, bacterial, yeast, and viral vectorsprepared, for example, from retroviruses, including lentiviruses,adenoviruses, adeno-associated viruses, and envelope-pseudotypedviruses. Vectors can be introduced into cells, for example, usingnanoparticles, hydrodynamic delivery, electroporation, sonoporation,calcium phosphate precipitation, or cationic polymers such asDEAE-dextran. Vectors can be complexed with lipids, such as encapsulatedin liposomes, or associated with cationic condensing agents.

ATF5 peptides of the invention can be delivered to cells via mechanismsthat exploit cellular receptors. Examples of such mechanisms includeantibody-drug conjugates, chimeric antigen receptors, andintegrin-targeting, RGD-like sequences. Examples of RGD-like sequencesinclude GRGD5 (SEQ ID NO: 72) and GRGDNP (SEQ ID NO: 73). ATF5 peptidesof the invention can comprise one or more RGD-like sequences, such astwo, three, four, or five RGD-like sequences, linked as described hereinor by any method known in the art. The one or more RGD-like sequence(s)can be incorporated to the N-terminal or C-terminal side of the ATF5leucine zipper region. Such RGD-like sequences can also be in retroinverso form, independently of one another and of the ATF5 leucinezipper region. Alternatively, ATF5 peptides can be encapsulated anddelivered to cells in vesicles, such as exosomes or liposomes, or inmicelles. Another method for introducing ATF5 peptides into cells is viacyclization, for example, using hydrocarbon staples (Bernal et al. 2007;Bird et al. 2016) or other cyclization methods known in the art.

Certain ATF5 peptides of the present invention comprise acell-penetrating domain or cell-penetrating peptide (CPP). The terms“cell-penetrating domain,” “cell-penetrating region,” and“cell-penetrating peptide” are used interchangeably herein.

CPPs are short (typically about 6-40 amino acids) peptides that are ableto cross cell membranes. Many CPPs are capable of crossing theblood-brain barrier (BBB). In some embodiments, the CPP is 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 amino acids in length,including ranges having any of those lengths as endpoints, for example,10-30 amino acids. CPPs have the ability to transport covalently ornon-covalently linked molecular cargo, such as polypeptides,polynucleotides, and nanoparticles, across cell membranes and the BBB.The translocation can be endocytotic or energy-independent (i.e.,non-endocytotic) via translocation. Numerous CPPs are described andcharacterized in the literature (see, e.g., Handbook of Cell-PenetratingPeptides (2d ed. Ulo Langel ed., 2007); Hervé et al. 2008; Heitz et al.2009; Munyendo et al. 2012; Zou et al. 2013; Krautwald et al. 2016). Acurated database of CPPs is maintained at crdd.osdd.net/raghava/cppsite(Gautam et al. 2012).

Peptides referred to as nuclear localization sequences (NLSs) are asubset of CPPs. The classical NLS contains one (monopartite) or two(bipartite) regions of basic amino acids. Consensus sequences ofclassical monopartite and bipartite NLSs are, respectively, K(K/R)X(K/R)(SEQ ID NO: 66) and (K/R)(K/R)X₁₀₋₁₂(K/R)_(3/5) (SEQ ID NO: 67), where3/5 indicates that at least 3 of 5 consecutive amino acids are lysine orarginine (Kosugi et al. 2009). An NLS sequence from SV40 large Tantigen, PKKKRKV (SEQ ID NO: 36), is an example of a classicalmonopartite NLS, while an NLS sequence from nucleoplasmin,KRPAATKKAGQAKKK (SEQ ID NO: 68) is an example of a classical bipartiteNLS (Lange et al. 2007; Kosugi et al. 2009). There are also numerousnon-classical NLSs, such as those from ribonucleoproteins (RNPs) hnRNPA1, hnRNP K, and U snRNP (Mattaj et al. 1998).

Non-limiting examples of CPPs suitable for use in the present inventioninclude peptides derived from proteins, such as from Drosophilaantennapedia transcription factor (Penetratin and its derivatives RL-16and EB1) (Derossi et al. 1998; Thorén et al. 2000; Lundberg et al. 2007;Alves et al. 2008); from HIV-1 trans-activator of transcription (Tat)(Vivés et al. 1997; Hällbrink et al. 2001); from rabies virusglycoprotein (RVG) (Kumar et al. 2007); from herpes simplex virus VP22(Elliott et al. 1997); from antimicrobial protegrin 1 (SynB) (Rousselleet al. 2001), from rat insulin 1 gene enhancer protein (pIS1) (Kilk etal. 2001; Magzoub et al. 2001); from murine vascular endothelialcadherein (pVEC) (Elmquist et al. 2001); from human calcitonin (hCT)(Schmidt et al. 1998); and from fibroblast growth factor 4 (FGF4) (Jo etal. 2005). CPPs suitable for use in the invention also include syntheticand chimeric peptides, such as Transportan (TP) and its derivatives(Pooga et al. 1998; Soomets et al. 2000); membrane translocatingsequences (MTSs) (Brodsky et al. 1998; Lindgren et al. 2000; Zhao et al.2001), such as the MPS peptide (also known as fusion sequence-basedpeptide or FBP) (Chaloin et al. 1998); sequence signal-based peptide(SBP) (Chaloin et al. 1997); model amphipathic peptide (MAP) (Oehlke etal. 1998; Scheller et al. 1999; Hällbrink et al. 2001), translocatingpeptide 2 (TP2) (Cruz et al. 2013), MPG (Morris et al. 1997; Kwon et al.2009), Pep-1 (Morris et al. 2001; Muñoz-Morris et al. 2007), andpoly-arginine (e.g., R₇-R₁₂) (SEQ ID NO: 87) (Mitchell et al. 2000;Wender et al. 2000; Futaki et al. 2001; Suzuki et al. 2002).Representative but non-limiting sequences are shown in Table 3.

TABLE 3 Peptide Sequence Bax-inhibiting VPTLK (SEQ ID NO: 69)peptide NLS1 Bax-inhibiting KLPVM (SEQ ID NO: 70) peptide NLS2 c-Myc NLSPAAKRVKLD (SEQ ID NO: 71) C. elegans SDC3 FKKFRKF (SEQ ID NO: 15)EB1 CPP LIRLWSHLIHIWFQNRRLKWKKK (SEQ ID NO: 16) FBP CPPGALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 17) FGF4 CPPAAVALLPAVLLALLAP (SEQ ID NO: 18) HATF3 ERKKRRRE (SEQ ID NO: 19) hCT CPPLGTYTQDFNKTFPQTAIGVGAP (SEQ ID NO: 20) MAP CPPKLALKLALKALKAALKLA (SEQ ID NO: 21) MPG CPPGLAFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 22) NF-κBVQRKRQKLMP (SEQ ID NO: 23) OCT-6 GRKRKKRT (SEQ ID NO: 24) Penetratin CPPRQIKIWFQNRRMKWKK (SEQ ID NO: 25) Penetratin CPPRQLKLWFQNRRMKWKK (SEQ ID NO: 26) variant 1 Penetratin CPPREIKIWFQNRRMKWKK (SEQ ID NO: 27) variant 2 Pep-1 CPPKETWWETWWTEWSQPKKRKV (SEQ ID NO: 28) pIsl CPPPVIRVWFQNKRCKDKK (SEQ ID NO: 29) Poly-Arg CPPRRRRRR(R)₁₋₆ (SEQ ID NO: 30) pVEC CPP LLIILRRRIRKQAHAH (SEQ ID NO: 31)RL-16 CPP RRLRRLLRRLLRRLRR (SEQ ID NO: 32) RVG CPPRVGRRRRRRRRR (SEQ ID NO: 33) R₆W₃ CPP RRWWRRWRR (SEQ ID NO: 34) SBP CPPMGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 35) SV40 PKKKRKV (SEQ ID NO: 36)SynB1 CPP RGGRLSYSRRRFSTSTGR (SEQ ID NO: 37) SynB3 CPPRRLSYSRRRF (SEQ ID NO: 38) SynB5 CPP RGGRLAYLRRRWAVLGR (SEQ ID NO: 39)Tat⁴⁷⁻⁵⁷ CPP YGRKKRRQRRR (SEQ ID NO: 40) Tat⁴⁷⁻⁵⁶ CPPYGRKKRRQRR (SEQ ID NO: 41) Tat⁴⁸⁻⁵⁶ CPP GRKKRRQRR (SEQ ID NO: 42)Tat⁴⁸⁻⁶⁰ CPP GRKKRRQRRRPPQ (SEQ ID NO: 43) TCF1-αGKKKKRKREKL (SEQ ID NO: 44) TFIIE-β SKKKKTKV (SEQ ID NO: 45) TP CPPGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 46) TP10 CPPAGYLLGKINLKALAALAKKIL (SEQ ID NO: 47) TP2 CPPPLIYLRLLRGQF (SEQ ID NO: 48) VP22 CPPDAATATRGRSAASRPTQRPRAPARSASRPRRPVQ (SEQ ID NO: 49)

Because the function of CPPs depends on their physical characteristicsrather than sequence-specific interactions, they can have the reversesequence and/or reverse chirality as those provided in Table 3 and/orknown in the art. For example, retro inverso forms of the CPPs (reversesequence and reverse chirality) are suitable for use in the invention.One example of a retro inverso CPP has the D-amino acid sequenceKKWKMRRNQFWIKIQR (SEQ ID NO: 50). Another example of a retro inverso CPPhas the D-amino acid sequence KKWKMRRNQFWLKLQR (SEQ ID NO: 51). Variantsof these sequences with one or more amino acid additions, deletions,and/or substitutions that retain the ability to cross cell membranesand/or the BBB are also suitable for use in the invention. The ATF5peptides of the invention can include a cell-penetrating domain havingat least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to the exemplary sequences provided in Table 3. The effectof the amino acid addition(s), deletion(s), and/or substitution(s) onthe ability of the CPP to mediate cell penetration can be tested usingmethods known in the art.

III. Methods of Preparation

ATF5 peptides of the invention can be chemically synthesized, forexample, using solid-phase peptide synthesis or solution-phase peptidesynthesis, or a combination of both. Synthesis may occur as fragments ofthe peptide which are subsequently combined either chemically orenzymatically. ATF5 polypeptides of the invention can be expressed usingrecombinant methods.

Accordingly, also provided are nucleic acid molecules encoding ATF5peptides of the invention. Such nucleic acids can be constructed bychemical synthesis using an oligonucleotide synthesizer. Nucleic acidmolecules of the invention can be designed based on the amino acidsequence of the desired ATF5 peptide and selection of those codons thatare favored in the host cell in which the recombinant ATF5 peptide willbe produced. Standard methods can be applied to synthesize a nucleicacid molecule encoding an ATF5 peptide of interest.

Once prepared, the nucleic acid encoding a particular ATF5 peptide canbe inserted into an expression vector and operably linked to anexpression control sequence appropriate for expression of the peptide ina desired host. In order to obtain high expression levels of the ATF5peptide, the nucleic acid can be operably linked to or associated withtranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

A wide variety of expression host/vector combinations can be employed toanyone known in the art. Useful expression vectors for eukaryotic hostsinclude, for example, vectors comprising expression control sequencesfrom SV40, bovine papilloma virus, adenovirus, and cytomegalovirus.Useful expression vectors for bacterial hosts include known bacterialplasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9and their derivatives, wider host range plasmids, such as M13, andfilamentous single-stranded DNA phages.

Suitable host cells include prokaryotes, yeast, insect, or highereukaryotic cells under the control of appropriate promoters. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells can be established or cell lines ofmammalian origin, examples of which include Pichia pastoris, 293 cells,COS-7 cells, L cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO)cells, HeLa cells, and BHK cells. Cell-free translation systems can alsobe employed.

EXAMPLES

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, can be practiced without departing from the scope of thepresent disclosure. A number of variants to reference peptide ST-3 weremade and examined, as described below.

Example 1. ST-3 Variants with Conserved Cysteine Substitution have InVitro Activity

The requirement of the single cysteine in ST-3 at amino acid position 21for cytotoxic activity was examined using a cytotoxicity assay in HL60cells. The cysteine residue is highly conserved within the native ATF5domain, and is thought to be required for homodimerization of ATF5 priorto DNA binding.

HL60 PML suspension cells were set at a density of 3.5×10³ cells/well in150 μL of RPMI+1.5% fetal bovine serum (FBS) in a 96 well dish. ST-3,reconstituted at a concentration of 10 mg/mL in 20 mM His, pH 7.5, wasadded to each well at a volume of 50 μL to a final concentration rangeof 0-80 μM. Cells were incubated with ST-3 for 48 hours at 37° C. Cellviability was quantified by flow cytometry using an Abcam Annexin V FITCapoptosis detection kit. Briefly, cells were washed with PBS andresuspended in 1× assay buffer containing Annexin V FITC and propidiumiodide (PI). Annexin V detects apoptotic cells, and PI stains deadcells. After staining, apoptotic cells show green fluorescence, deadcells show red and green fluorescence, and live cells show little or nofluorescence. Cells were selected for analysis based on forward scatter(FSC) vs. side scatter (SSC), and analyzed by BD Accuri C6 Plus flowcytometer to detect Annexin V-FITC binding (Ex=488 nm; Em=530 nm) usingFITC signal detector and PI staining by the phycoerythrin emissionsignal detector. Percentage of Annexin V^(low) and PI^(low) werequantified and presented as % Viability.

Lyophilized ST-3 or ST-3 with glycine (ST-4) or alanine (ST-5)substituted for cysteine at amino acid position 21 was freshlyreconstituted in histidine buffer to a stock solution of 5 mg/mL andadded to cells at a final concentration range of 0-40 μM. Cells wereincubated with ATF5 peptide for 48 hours prior to quantification of cellviability. Results are shown in FIG. 2.

ST-3 activity is attenuated but not ablated by substitution of cysteineat position 21 with alanine or glycine. Observed EC₅₀ values wereapproximately 16 μM, 35 μM, and 38 μM for ST-3, ST-4, and ST-5,respectively. In functional assays, EC₅₀ is the concentration thatreduces a biological response by 50% of its maximum. In the case of ATF5peptides, EC₅₀ is measured as the concentration that reduces cellviability by 50% of its maximum. EC₅₀ can be calculated by any number ofmeans known in the art. Substitution of alanine for cysteine did notimpact cell penetration. Penetration of the glycine-substituted variantwas not tested; however, based on data for alanine substitution, cellpenetration is not expected to be affected.

Example 2. ST-3 Variants with Non-Conserved Substitutions have In VitroActivity

The ST-3 variants discussed in this Example are summarized in Table 4.ST-3 has an EC₅₀ of about 12-20 μM.

TABLE 4 EC₅₀ Variant Modification(s) (μM) ST-7 (SEQ ID NO: 7) N28L 3.1ST-8 (SEQ ID NO: 8) Q22A 5.4 ST-6 (SEQ ID NO: 6) S37A 11.2 ST-9 (SEQ IDNO: 9) E20R, E25R, R27E, K32E 2.0 ST-10 (SEQ ID NO: 10) E20R, E25R,R27E, N28L, 2.2 K32E, S37A ST-11 (SEQ ID NO: 11) E20R, C21A, E25R, R27E,4.2 N28L, K32E, S37A

Single Substitutions

Using the HL60 cell viability assay described in Example 1, we examinedcytotoxic activity of ST-3 variants containing single non-conservativeamino acid substitutions (FIG. 3). Variant ST-7 has a substitution ofthe polar asparagine with the non-polar leucine at position 28 and anEC₅₀ of approximately 3 μM. ST-8 has a substitution of the polarglutamine with the non-polar alanine at position 22 and an EC₅₀ ofapproximately 5 μM. This increase in activity for ST-7 and ST-8 is3-6-fold higher than that of ST-3. ST-6 has a conservative substitutionof serine with alanine at position 37 and comparable activity to ST-3.

Multiple Substitutions

Using the HL60 cell viability assay described in Example 1, we examinedcytotoxic activity of ST-3 variants containing multiple non-conservativeamino acid substitutions. Variant ST-9, has two negatively chargedglutamic acid residues that were each replaced with a positively chargedarginine, and a positively charged arginine and a positively chargedlysine that were each replaced with a negatively charged glutamic acid.(See Table 4.) These changes involved about 18% of the ATF5 leucinezipper region of ST-3. The activity of ST-9 was significantly increasedover that of ST-3 (FIG. 4). ST-9 has an EC₅₀ of approximately 2 comparedwith about 12-20 μM for ST-3.

ST-10 has the same four “charge” substitutions as ST-9, along with anon-conservative substitution of asparagine (polar) with leucine(non-polar) at position 28, as in ST-7, and a conservative substitutionof serine with alanine, as in ST-6. (See Table 4.) The activity of ST-10is comparable to that of ST-9 (FIG. 4).

ST-11 has the same substitutions as ST-10, plus a substitution ofalanine for the conserved cysteine at position 21, as in ST-5. (SeeTable 4.) ST-11 has improved cytotoxic activity over ST-3 (FIG. 4).

Example 3. Retro Inverso ST-3 Variants have In Vitro Activity

We examined the cytotoxicity of a retro inverso form of ST-11 in theHL60 cell viability assay described in Example 1. The retro inversovariant, ST-13, had comparable activity to ST-11, and superior activityto ST-3 (FIG. 5).

ST-13 was further examined in various human cancer cell lines, as was asecond retro-inverso variant, ST-14. Suspension cells (HL60, AML14, orSET2) were set at a density of 3.5×10³ cells/well in 150 μL of RPMI+1.5%fetal bovine serum (FBS) in a tissue culture-treated 96 well dish. ATF5peptide, reconstituted at a concentration of 10 mg/mL in 20 mM His, pH7.5, was added to each well at a volume of 50 μL to a finalconcentration range of 0-80 μM. Cells were incubated with peptide for 48hours at 37° C. Cell viability was quantified by flow cytometry using anAbcam Annexin V FITC apoptosis detection kit. Briefly, cells were washedwith PBS and resuspended in 1× assay buffer containing Annexin V FITCand propidium iodide (PI). Cells were analyzed by BD Accuri C6 Plus flowcytometer to detect Annexin V-FITC binding (Ex=488 nm; Em=530 nm) usingFITC signal detector, and to detect PI staining using the phycoerythrinemission signal detector. Percentage of Annexin V^(low) and PI^(low)were quantified and presented as % Viability. EC₅₀ values werecalculated using GraphPad Prism v.7 XML.

Additionally, adherent A375, MCF7, U251, DU145, U87, and A549 cells wereset at a density of 3.5×10³ cells/well in 200 μL of media on day −1. Onday 0, media was removed and replenished with 150 μL of fresh media, andcells were treated with ATF5 peptide as described. Following a 48-hourincubation at 37° C., floating cells were collected, adherent cells werewashed with Dulbecco's Phosphate Buffered Saline (DPBS), dissociatedfrom the dish with 50 μL 2.5% trypsin at RT, and combined with floatingcells. Cell viability was determined as described above for suspensioncells or by MTT assay (MCF7 cells). Alternatively, cells were gentlyremoved from the cell culture plate following trypsinization with 2.5%trypsin, and cell viability was quantified by flow cytometry using anAbcam Annexin V FITC apoptosis detection kit, as described above.

PBMC and BMIVIC were cultured under the conditions described above forsuspension cells.

Both ST-13 (FIGS. 6A-6E) and ST-14 (FIGS. 7A-7E; Table 5) showedsignificant cytotoxic activity in a wide range of tumor cell types;ST-14 was also cytotoxic in peripheral blood mononuclear cells (PBMC)and bone marrow mononuclear cells (BMMC) (Table 5).

TABLE 5 Cell Line ST-14 Calculated EC₅₀ (μM) HL60 9.2 AML14 4.8 A375 0.7MCF7 2.1 U251 2.2 U87 3.6 DU145 4.0 A549 1.2 SET2 22.1 PBMC >80 BMMC >80

We measured RNA expression of ATF5 and apoptosis regulatory proteins,Mcl-1, Bcl-2, and Survivin, relative to (3-actin expression in HL60cells following treatment with ST-14. Briefly, 8×10⁵ HL60 suspensioncells were plated in a 6-well plate and treated with 0 μM, 5 μM, 20 μM,or 40 μM ST101 for 4 or 24 hours. Cells were centrifuged at 1,200 rpmfor 7.5 minutes, and pellets were washed with 750 μM DNAse-RNAse-freeH₂O to remove residual media. RNA was extracted using Quick-RNA™MiniPrep Plus kit (Zymo Research Cat. #R1054) according to themanufacturer's instructions and eluted with 55 μM H₂O. RNA (200 ng) wasrun on Invitrogen pre-cast 2% SYBR™ Gold E-Gel (Thermo Fisher ScientificCat. #G401002) to assess RNA quality. cDNA was synthesized usingInvitrogen SuperScript™ IV VILO Master Mix with ezDNAse™ Enzyme (ThermoFisher Scientific Cat. #11766050) according to manufacturer's protocolwith the following modification: the cDNA was extended at 56° C. insteadof 50° C. Minus RT controls were set up to rule out gDNA contamination.cDNA was amplified using gene-specific primers (Table 6) and KOD Xtreme™Hot Start DNA Polymerase (Millipore Sigma Cat. #71975-3). An equalamount of RNA was added to each reaction. All PCR products were run onInvitrogen pre-cast 2% ethidium bromide E-Gel (Thermo FisherScientific). Gene expression was compared to β-actin using BioRadImageLab software.

TABLE 6 Gene Primer Sequence SEQ ID NO β-Actin hsACTB_FATG GAT GAT GAT ATC GCC GCG C 74 β-Actin hsACTB_RGAA GCA TTT GCG GTG GAC GAT G 75 Bcl2α, β hsBcl2 alpha beta_FATG GCG CAC GCT GGG 76 Bcl2α hsBcl2 alpha_R CTT GTG GCC CAG ATA GGC ACC77 Bcl2β hsBcl2 beta_R GCC CAG ACT CAC ATC ACC AAG TG 78 Mcl1 hsMcl1_FATG TTT GGC CTC AAA AGA AAC GCG G 79 Mcl1 hsMcl1_RTCT TAT TAG ATA TGC CAA ACC AGC 80 TCC TAC TCC ATF5 hsATF5_FATG TCA CTC CTG GCG ACC CT 81 ATF5 hsATF5_R GCA GCT ACG GGT CCT CTG G 82CEP13β hsCEBP beta_F ATG CAC CTG CAG CCC G 83 CEP13β hsCEBP beta_RCGC GCA GTT GCC CAT GG 84 Birc5 hsBIRC5_F ATG GGT GCC CCG ACG TTG 85Birc5 hsBIRC5_R TCA ATC CAT GGC AGC CAG CTG 86

ST-14 exposure resulted in reduced expression of Mcl-1, Bcl2, and BIRC5(Survivin), relative to (3-actin expression (FIGS. 8A-8D).

Example 4. ST-3 Variants have In Vivo Activity

We examined the effect of ST-3 and three variants on tumor volume in aHL60 subcutaneous tumor model. Briefly, 5×10⁶ HL60 cells, suspended 1:1in Matrigel, were implanted via subcutaneous injection into the axillaof NU/J mice. Dosing was initiated on day 2 post tumor inoculation, withaverage tumor volume ranging from 144-176 mm³. ATF5 peptides wereadministered at a dose of 25 mg/kg via intraperitoneal (IP) injectiontwice a day. All of the tested variants reduced tumor volume relative tovehicle, with the retro inverso variant, ST-13, showing the greatestanti-tumor activity (FIG. 9).

We examined the effect of ST-14 on tumor volume in tumor models usingU251 glioblastoma cells, MCF7 breast cancer cells, HL60 promyelocyticleukemia cells, and A375 melanoma cells. Briefly, 5×10⁶ cells, suspended1:1 in Matrigel, were implanted via subcutaneous injection into theaxilla of NU/J mice (for MCF7 and HL60 cells) or NOD/SCID mice (for U251and A375 cells).

For U251 cell tumors, dosing was initiated on day 2 post tumorinoculation, with average tumor volume of about 240 mm³. ST-14 wasadministered at a dose of 50 mg/kg via subcutaneous (SC) injection threetimes per week for three weeks. ST-14 significantly reduced tumor volume(FIGS. 10A, 10C) and increased survival (FIGS. 10B-10C) relative tovehicle. Similar results were achieved with a dose of 25 mg/kg ST-14.

For MCF7 cell tumors, we examined the effect of immediate dosing anddelayed dosing. In the immediate dosing experiment, dosing was initiatedon day 2 post tumor inoculation (2×10⁶ cells), with average tumor volumeof about 280-330 mm³. ST-14 was administered at a dose of 25 mg/kg viaSC injection three times per week for three weeks. In the delayed dosingexperiment, dosing was initiated on day 59 post tumor inoculation(vehicle: 2×10⁶ cells; treatment group: 5×10⁶ cells). Tumor volume wasmonitored for 92 days post-inoculation. In both the immediate anddelayed treatment groups, ST-14 significantly reduced tumor volumerelative to vehicle for the duration of monitoring (FIGS. 11A-11B).

For HL60 cell tumors, dosing was initiated on day 2 post tumorinoculation, with average tumor volume of about 220 mm³. ST-14 wasadministered at a dose of 20 mg/kg via SC injection three times per weekfor three weeks. ST-14 significantly reduced tumor volume relative tovehicle (FIG. 12).

For A375 cell tumors, dosing was initiated on day 2 post tumorinoculation, with average tumor volume of about 250-344 mm³. ST-14 wasadministered at a dose of 25 mg/kg via SC injection twice daily forthree weeks. ST-14 significantly reduced tumor volume relative tovehicle (FIG. 13).

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The present invention is further described by the following claims.

1. An ATF5 peptide comprising a truncated ATF5 leucine zipper region,wherein the truncated ATF5 leucine zipper region has a D-amino acidsequence (SEQ ID NO: 65) VAEAREELERLEARLGQARGEL.


2. The ATF5 peptide according to claim 1 comprising a D-amino acidsequence (SEQ ID NO: 14) VAEAREELERLEARLGQARGELKKWKMRRNQFWLKLQR,

wherein the ATF5 peptide is a cell-penetrating peptide.
 3. The ATF5peptide according to claim 1, wherein the peptide does not comprise anextended leucine zipper region.
 4. The ATF5 peptide according to claim1, wherein the peptide comprises an N-terminal acetyl group and/or aC-terminal amide group.
 5. A composition comprising the ATF5 peptideaccording to claim
 1. 6. The composition according to claim 5, which isa pharmaceutical composition.
 7. A kit comprising the ATF5 peptideaccording to claim
 1. 8. A nucleic acid molecule encoding the ATF5peptide according to claim
 1. 9. A method of promoting cytotoxicity in aneoplastic cell, the method comprising contacting the neoplastic cellwith the ATF5 peptide according to claim
 1. 10. A method of inhibitingproliferation of a neoplastic cell, the method comprising contacting theneoplastic cell with the ATF5 peptide according to claim 1.